Quantitative Determination of Acetylcholine and Choline in Microdialysis Samples by MALDI-TOF MS

Abstract

A simple, sensitive, and fast matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) method was developed for the determination of acetylcholine (ACh) and choline (Ch) in microdialysis samples. An optimized dried droplet preparation with the common matrix alpha-cyano-4-hydroxycinnamic acid (CHCA) was used. Limits of detection [signal-to-noise ratio (S/N) = 3] of 0.3 fmol/microL for ACh and 20 fmol/microL for Ch were found using standards diluted in artificial cerebrospinal fluid (aCSF). The limit of quantification (LOQ) for ACh was 1 fmol/microL, and excellent linearity (R(2) = 0.9996) was maintained over the range of 1-1000 fmol/microL. Choline was quantified over the range of 0.1-50 pmol/microL, also with excellent linearity (R(2) = 0.9995). Good accuracy and precision were obtained for all concentrations within the range of the standard curve. The developed method was successfully used for the determination of ACh and Ch in mouse brain microdialysis samples. The samples were quantified by a calibration curve and also by the method of standard addition. Despite the high salt content of the perfusion fluid (>150 mM), a direct measurement was possible. To the authors' knowledge, this is the first published method to determine acetylcholine and choline in microdialysis samples by MALDI-TOF MS. The method presented significantly reduces the needed analysis time, as only approximately 10 s/sample is required, and it is also possible to improve the temporal resolution, because only approximately 1 microL of sample is needed.

High Throughput Enzyme Inhibitor Screening by Functionalized Magnetic Carbonaceous Microspheres and Graphene Oxide-Based MALDI-TOF-MS

“…Suggested by others [12], the best options for internal standards are isotopically labeled standards, which are expensive and frequently unavailable. To address this problem, structurally analogous compounds are frequently utilized as a substitution.…”

“…ACh has been detected by various methods, such as liquid chromatography/electrochemical (LC-EC) method [36][37][38], liquid chromatography/electrospray ionization/ mass spectrometry (LC-ESI-MS) [8,[39][40][41][42], liquid chromatography/atmospheric pressure chemical ionization/mass spectrometry (LC-APCI-MS) [43], as well as MALDI-TOF-MS with CHCA as the matrix [12]. The performances of these methods have been listed in detail on Table 3.…”

“…However, the performances of these matrices are not desirable when utilized to detect low molecular weight compounds (G600 Da) [35]. Strong background interferences were observed when CHCA was used as matrix [17], i.e., ACh and choline detection [12]. These interferences would greatly obscure the targeted compounds' detection.…”

“…No obvious changes of UV signal were observed of the supernatant solution. Due to the high sensitivity of the MALDI MS, a certain [12] hydrolyzing results were observed (the result is shown in Supporting Information, Figure S3). We assumed that as there existed lots of hydroxyl groups and carboxyl groups on the beads without GLYMO, the electrostatic interactions would possibly occur between the beads and amino acids of the enzyme.…”

“…However, two factors severely limit the application range of MALDI-TOF-MS, which are ion signal fluctuation produced by single laser-shot as well as intense matrix background interference. From reports by others [12], the introduction of internal standards can compensate signal variations successfully. On the other hand, background interferences could be greatly reduced by utilizing proper matrices (i.e., graphene), especially for small organic molecule detection (G600 Da) [13][14][15][16][17][18].…”